Project Summary Alcohol Use Disorders (AUDs) are highly prevalent among adults and adolescents worldwide, and pose a significant public health problem. Furthermore, they are highly heritable, and remain largely untreated. This project aims to identify cellular and molecular mechanisms underlying AUD development through the investigation of KCNJ6 upregulation in human neurons. Multiple single nucleotide polymorphisms (SNPs) in KCNJ6 have a genome wide association with an AUD risk electroencephalogram endophenotype. Specifically, SNPs associated with a reduction in frontal theta event-related oscillations in measures of attention and inhibitory control are also associated with an upregulation of KCNJ6 mRNA in the frontal cortex. KCNJ6 encodes the G protein-activated inwardly rectifying potassium channel 2 (GIRK2), a protein contributing to the maintenance of inhibitory tone in the brain. Therefore, the first aim is to elucidate functional effects of upregulated KCNJ6 expression in neurons derived from control human induced pluripotent stem cells. This approach capitalizes on the translational potential of hiPSC-derived neurons and minimizes the confounds of inter-individual variability by comparing endogenous and elevated KCNJ6 expression in neurons with the same genetic background. Calcium imaging and patch clamp electrophysiology will be used to measure changes in neuronal activity associated with elevated GIRK2, such as decreased spontaneous activity and excitability. The second aim is to test the effects of acute and chronic ethanol exposure on these neural cultures using calcium imaging and RNA sequencing analyses of differential gene expression. Alcohol directly activates GIRK2 channels, and elevated levels of the protein could result in increased neuronal inhibition in response to alcohol exposure. Furthermore, exposure to alcohol and changes in neuronal activity may impact global gene expression as measured by RNAseq, supporting the hypothesis that elevated KCNJ6 impacts transcriptomic adaptations to alcohol treatment. Overall, this project provides a multifaceted characterization of human neurons in an etiologically-relevant model of alcohol use disorder, potentially identifying mechanisms suitable for therapeutic targeting.